Methods and compositions for maintaining the conformation and structural integrity of biomolecules

ABSTRACT

A composition includes a target pharmaceutical or biological agent, a solution containing the target pharmaceutical or biological agent, and substrate that is soluble in the solution. The substrate is capable of being solidified via a solidification process and the solidification process causes the substrate to become physically or chemically cross-linked, vitrified, or crystallized. As a result of the solidification process, particles are formed. The target pharmaceutical or biological agent within the solution retains proper conformation to ultimately produce a desired effect.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/653,414 filed on Jul. 18, 2017, which claims priority to andincorporates by reference the entire disclosure of U.S. ProvisionalPatent Application No. 62/363,593, which was filed on Jul. 18, 2016.

FIELD OF THE INVENTION

This application relates to protecting pharmaceutical or biologicalmolecules from organic solvents and more particularly, but not by way oflimitation, to protecting molecules whose function is dependent onprimary, secondary, tertiary, and/or quaternary structure to achieve andmaintain function. This invention applies to the fields ofpharmaceutics, medical devices, drug delivery devices, tissueengineering, advanced or chronic wound healing, textiles, 2D and 3Dprinting, mesofabrication, fermentation, biotechnology, proteinproduction, genetics, genomics, protiomics, metabolomics, and the like.

BACKGROUND

U.S. Pat. No. 6,596,296 provides for “a composition of at least onebiodegradable polymer fiber wherein the fiber is composed of a firstphase and a second phase, the first and second phases being immiscible,and wherein the second phase comprises one or more therapeutic agents.”The immiscible phases allow for therapeutic agents incorporated into thecomposition to be partially protected from an adverse organicenvironment so as to maintain their biological function. However, theinterface between the phases can be detrimental to the therapeutic agentand alter its function. Therefore, this invention proposes animprovement over and an expansion of this concept to allow for theprotection of therapeutic agents in potentially damaging organic solventenvironments.

SUMMARY

In some embodiments, a composition includes a solution containing atarget pharmaceutical or biological agent, and a substrate that issoluble in the solution. The substrate is capable of being solidifiedvia a solidification process and the solidification process causes thesubstance to become physically or chemically cross-linked, vitrified, orcrystallized. During the solidification process, particles are formedcontaining the pharmaceutical or biological agent. The targetpharmaceutical or biological agent is located within the particles andretains proper conformation to ultimately produce a desired effect. Thesolidified particles range from being completely non-swellable to poorlyswellable in a solvent from which protection is desired. The term“poorly swellable” refers to a particle that shows an increase in sizerelative to a non-swellable particle when placed in a solvent from whichprotection is desired.

In some embodiments, the substrate is capable of solidification, and maybe in part or whole composed of various types of molecules: protein,carbohydrate, or synthetically derived molecules. The protein may befrom the families of gelatins, collagens, or fibrins. The carbohydratemay be from the families of monosaccharides, disaccharides,oligosaccharides, and polysaccharides, with illustrative examples toinclude sucrose, trehalose, maltose, dextran, starch, alginates,xanthan, galactomanin, agar, or agarose. The synthetically derivedmolecule may be from the families of poly(ethylene glycol), poloxamer orpolyesters. In some embodiments, the substrate may also includesubstances which do not inherently solidify, but may be needed tostabilize or aid the solidification process such as surfactants,stabilizers, emulsifiers, lyoprotectants, and cryoprotectants. Otherprocessing aids may be helpful as well, for example in one embodimentcholesterol is used. Other types of component molecules that may beincluded in the substrate are agents that are intended to initiateand/or propagate and/or terminate the solidification process. In someembodiments, the agent is either slow initiating or may be externallyinitiated by means such as light irradiation, temperature, mechanical,pH change, etc.

In some embodiments, a mean hydrodynamic diameter of the particles, whenin a solidified form, is less than 1 μm. In some embodiments, a meanhydrodynamic diameter of the particles, when in a solidified form, isgreater than 1 μm.

In some embodiments, the particles may be either inherently or with helpof an agent (i.e., surfactant, stabilizer, or emulsifier), mixed, orsuspended within the solvent from which protection is desired. In someembodiments, the particles are incorporated into a polymer solution. Insome embodiments, the polymer solution is extruded to create a fiberthat is loaded with the target pharmaceutical or biological agent. Insome embodiments, the polymer solution is three-dimensionally printed.In some embodiments, the surfactant, stabilizer, and emulsifier areincorporated into the substrate prior to solidification. In someembodiments, the surfactant, stabilizer, or emulsifier is incorporatedwith the substrate during or after solidification.

In some embodiments, the substrate is a mucopolysaccharide or a branchedglucan.

In some embodiments, the substrate being solidified is a therapeuticprotein and the solidification of the therapeutic protein alone providesprotection from the solvent.

In some embodiments, the solidification process can proceedspontaneously through temperature-induced phase change. In someembodiments, the solidification process can proceed through addition ofa cross-linking agent or other chemical entities that will ultimatelyinitiate, propagate or otherwise aid in the solidification process, andwherein the cross-linking agent may be internally added to the solutionthat will become a dispersed phase of an emulsion once formed.

In other embodiments, the solidification process may occur because ofdehydration, vitrification or crystallization of substrate that entrapsthe pharmaceutical or biological agent. Dehydration may occur at roomtemperature, elevated temperature or depressed temperature and in somecases, may be accompanied by vacuum applied to the suspension ormixture. In some cases, the dehydration may happen during alyophilization process, where sublimation is used to remove some or allliquid components of the mixture or suspension, leaving behind thepharmaceutical or biological agent within lyophilized substrate. Forliquid components of the mixture or suspension whose freezing point isbelow the capability of the lyophilization machine, these componentswill largely be removed by evaporation rather than sublimation.

DESCRIPTION

In this invention the terms “drug”, “agent”, “therapeutic agent”,“biologically active agent” are collectively and synonymously defined tobe compounds that based on structure or composition are expected to a)have physiological impact when introduced into a living organism,including human; or b) to act to catalyze or promote specific reactionsthat may or may not take place in a biological environment; for example,using enzymes in a non-biological environment to promote specific chiralchemistry. These compounds may be synthetically produced, or may be ofbiological origin, including by way of example: cells, viruses,proteins, peptides, oligonucleotides, all varieties of RNA and DNA,carbohydrates, lipids, etc.

There are numerous fields of medicine, pharmacology, as well as fieldssuch as biotechnology, where the preservation of nativeconfirmation/biological activity of an enzyme, therapeutic agent, ordrug must be maintained during processes that involve exposure toorganic solvents that might otherwise result in a decrease in theiractivity. To satisfy the need to preserve the potential activity ofthese therapeutic agents or drugs during the time when they are exposedto these solvents, a protective material may be used to isolate thedrugs or therapeutic agents from this organic solvent environmentwithout physically or chemically altering the agent itself. Thisprotective material containing the drug may be mixed or suspended intothe organic solvent of interest. There are numerous examples where thisinvention is needed; these are set forth as illustrative, and not meantto be comprehensive.

Fiber manufacturing using wet extrusion to create a drug-loaded fiber,textile or similar biomedical structure, is an example where, over theduration of the extrusion and until processes designed to removeresidual solvent are completed, the drug must be protected from thesolvent systems involved in the extrusion process. In this application,the choice of the materials used, and the choice of which type ofcondensation, gelling, or solidification method applied will alsocontribute to the rate at which drug is released from the fiber once thebiomedical structure or device is in use.

Three-dimensional printing is another area where preservation of thenative structure of a drug is desired. Similar to solvent exposureduring an extrusion process, the drug must be protected while it is inthe liquid (ink) form. This may require extremely long-term stability ofthe drug in solution, with months to even years of stability likelyrequired in this application.

This invention relates to the composition of a protection matrix thatmay be used to encapsulate molecules that are sensitive to organicsolvent exposure, wherein the encapsulating matrix mitigates damage tothese molecules. There are numerous different materials that can be usedas a protective matrix, including, in some cases, the drug itself. Theessence of this invention is to identify the fundamental rules that mustbe followed to ensure that the drug of interest is likely to beprotected. This invention also relates to the creation of aself-protection conformation for a given drug, wherein the drug isself-encapsulated and protected from the organic solvent. Some proteins,for example, can be reversibly precipitated to provide shielding fromorganic solvents.

The fundamental rules are as follows: The composition of substrate thatis used to protect the drugs of interest must be soluble in a solutionwith the drugs to be protected. This solution may be composed of anyfluids, salts, surfactants or stabilizers needed for the molecule ofinterest. It may also contain other additives as may be needed forprocessing steps, as will be illustrated in the various embodiments andexamples to follow. This fluid must maintain the conformation of thedrugs of interest or allow recovery to the conformation so that they caneventually perform their desired function.

The composition of substrate that is used to protect the drugs ofinterest must include components that are able to be cross-linked,gelled, vitrified, crystalized or by some means formed into particlesthat entrap or in some way contain and protect the drugs of interest.

The resulting particles used to protect the drugs of interest range fromnon-swellable to poorly swellable in the fluid from which protection issought.

Any set of materials that meets all the above three rules or conditionsand that is used for the specific purpose of protecting drugs from thepresence of organic solvents is the topic of this invention. To meetmost industrial and academic needs, it is usually desired that theparticles are in the tens to thousands of nanometers diameter range.

As a practical matter of reduction to practice, it is usually requiredthat the particles are suspendable within the organic solvent from whichprotection is sought. This ability to suspend these (typically nano-)particles is often achieved by associated surfactants. Suspension withinthe organic solvent of interest can also be achieved by particleinteractions or the lack thereof (neutral surfaces). Surface surfactantsprovide sufficient organic interface to suspend these particles, and inmany embodiments, reduce the aggregation potential of the particles.This typically results in a colloidal suspension, often visiblyappearing as an opalescent suspension.

As this invention has been reduced to practice, there are manyembodiments that will be illustrative of the process and teach thepractice of creating these protection matrices. The embodiments providedwithin this specification are meant only to be illustrative, and do notrepresent an exhaustive list of applications of this invention.

In one embodiment, a water-in-oil micro or nanoemulsion is used toensure that created particles are of a desired diameter, which isderived from the size of the dispersed aqueous phase in the emulsion. Inthis embodiment, a water phase of the emulsion contains theencapsulating substrate, the molecules to be protected, and any salts orstabilizers required by the molecules to be protected. The aqueous phasemay often include a “water-side” surfactant to help stabilize theemulsion. If the encapsulating material performs its function ofcondensing, cross-linking, gelling, etc. as a result of a chemicalreaction, that initiating chemical may also be present in the aqueoussolution, potentially in an inactive state, or with a built-in delay.The oil phase of the emulsion will generally contain one or moresurfactants. The oil phase may also contain chemicals that can act toinitiate and/or propagate the condensing, cross-linking or gelling ofthe encapsulating material. Generally, the oil phase is chosen basedupon the criteria of ease of removal and isolation of the formedencapsulating particles.

As a specific example of this embodiment, the molecule to beencapsulated is a therapeutic protein. The encapsulating material isgelatin, which will form a thermally-reversible “physical gel” when asolution of appropriate concentration is cooled. The organic phase iscyclohexane containing Span 80 (Sorbitan Monooleate). The aqueous phasecontains the protein to be protected, Tween 80 (Polysorbate 80), and anysalts or other stabilizers required of the protein. With appropriateratios of Span 80, Tween 80, and organic to aqueous phases, a stablemicro-emulsion or nano-emulsion can be formed. The aqueous phase in thisemulsion consists of stable, isolated, dispersed droplets of water whosemean diameter is generally in the nanometer size. As gelatin thermallyforms a gel upon cooling, the entire emulsion is slowly cooled. As thegelatin in each nano-droplet of water cools, it will begin to condense,thereby trapping the therapeutic protein and excess Tween 80 within eachforming nanogel. As the cooling continues, the cyclohexane organic phasefreezes. The cooling rate can then be dramatically accelerated toquickly freeze the water in the aqueous phase. Once all components arefrozen, the entire emulsion can be lyophilized to remove both thecyclohexane as well as the water, ultimately leaving only the gelatinnanogels containing the therapeutic protein and surfactants.

This same system can use other materials as well, for example sodiumalginate can be substituted for gelatin. In this embodiment, CaCl₂ isadded to the cyclohexane. There is enough solubility of the CaCl₂ inthis environment that the Ca⁺² ions are able to disperse throughout theemulsion, providing the ability to cross-link the alginate in thedispersed water phase.

The above two examples of thermal and chemical gelation serve asillustrations of reduction to practice of this embodiment ofrequirements one and two of this invention, i.e. that the encapsulatingmaterial (gelatin or alginate in the above examples) is soluble in theaqueous phase with the therapeutic protein as well as other moleculesneeded for processing, such as Tween 80. The second requirement is thatthe encapsulating material must be able to condense, gel, cross-link,vitrify, etc. The above two examples demonstrate two differentmechanisms whereby this may take place. The third rule is that theencapsulating material must be very poorly swellable in the organicphase from which protection of the therapeutic protein is sought. Thisexample illustrates how this concept can be tailor made for eachsituation. For example, gelatin is swellable and soluble in 1,1,1-3,3,3hexafluoro-2 propanol, (HFIP) but not in dichloromethane (DCM).Therefore, if the solvent from protection is sought is HFIP, gelatin isa very poor choice, as the solvent will easily swell and penetrate thegelatin matrix and have full access to the therapeutic agent ofinterest, and thus violate rule three. However, if the solvent fromwhich protection is sought is dichloromethane, gelatin is nearlycompletely insoluble in this material and will not significantly swell,nor will the solvent penetrate into the gelatin nanogels, therebyprotecting the therapeutic protein of interest.

The above examples illustrate that if one knows which solvent(s) themolecules will be exposed to, it is possible, and very straight forward,to use the rules of this invention to design a system that will protectthe molecules of interest.

WORKING EXAMPLES Example 1

Dissolve 5% w/v gelatin in 10 mL Type I water in a scintillation vial bymagnetically stirring at 37° C. for 1 hour. Weigh out a 3:1 mass/massmixture of Span 80 and Tween 80 into a scintillation vial. When thegelatin has completely dissolved, add 1 mL of this warm solution to avial containing lyophilized growth factor and allow to dissolve.Maintain at 37° C. Add 15 mL of dry cyclohexane to the scintillationvial. Vortex the scintillation vial to incorporate the surfactants intothe cyclohexane. Place a vial into the chilled water bath and, whilesonicating, slowly add the gelatin solution/growth factor drop-by-dropusing a pipette. When complete, immediately place the vessel into anenvironmental chamber at 8° C. and set the mixer plate to rotate at aspeed of 35/70 for a minimum of 60 minutes to gel the gelatin. Awater-soluble, non-toxic cross-linking system, such asN-hydroxysuccinimide (NHS) and1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (CDI) canbe incorporated into the process to further condense and solidify thegelatin nanoparticles. Snap freeze and lyophilize the solution at thecompletion of gelation.

Example 2

Substitute sodium alginate for gelatin in Example 1 and add measuredquantity of CaCl₂ to the formed emulsion to yield a 40 mM solution withrespect to the aqueous portion in order to gel the alginate. The NHS andCDI are also omitted. Freeze and lyophilize the emulsion followinggelation of the alginate.

Example 3

In a scintillation vial, create a dextran solution in type I water at 6%w/vol. In another scintillation vial, weigh an appropriate mass of Tween80 to yield a solution at 0.058 g/ml in the dextran solution. Mixappropriately. Weigh out lysozyme to be dissolved in 3.0 mL of thedextran/T80 solution to yield the final desired concentration. Transferthe appropriate volume into the scintillation vial containing lysozymeto create a 0.003 g/mL solution. Weight Span 80 into a 45 mLscintillation vial. Add an appropriate volume of filtered cyclohexane tothe vial to obtain a final solution concentration of 0.0118 g/mL of thedesired volume. Vortex to incorporate the surfactant. Place thecyclohexane/Span vial into a chilled water bath and immerse a sonicatorprobe. While sonicating, add the desired volume of the dextran 70/Tween80/lysozyme solution to the cyclohexane/Span 80, drop-by-drop, toemulsify. Place the vial into a freezer at −20° C. and allow to slowfreeze for a minimum of four hours, preferably overnight, to freeze thefree-water in the dextran/lysozyme dispersed aqueous phase. Lyophilizethe frozen emulsion upon completion.

Example 4

To a measured quantity of lysozyme, add 0.020 g of poloxamer P188surfactant. Solubilize in Type I water at approximately 75 mg/mL. Add0.96 g of PLGA to a tall scintillation vial and dissolve with 16 mL offiltered acetone. Maintain vessel temperature at 25° C. While stirring,add 500 μL aliquots of acetone slowly into the lysozyme solution until2.5 mL have been added. Cap the vessel and hold for 60 seconds. Repeatthe additions until approximately 5 mL have been added to the vessel.Cap and hold another 60 seconds. Switch to 100 μL aliquots of acetoneand add while stirring. Cap the vessel and hold between additions for 30seconds. Continue to add acetone until the solution turns and remainsopalescent, indicating the protein has nanoprecipitated. Cap the vesseland stir for five minutes. Add a stir bar to the polymer solution andadd the nanoprecipitate solution into the polymer solution in smallaliquots. When complete, stir for an additional 5 minutes. The resultantsolution should be opalescent and stable. Place a beaker containing 500mL of pentane into a sonic water bath. Syringe deliver the polymersolution into the pentane bath, while sonicating, via a small diameterblunt needle at no more than 0.5 mL/min. When delivery is complete,allow the material to sit statically in pentane for 30 minutes tosolidify the polymer. Pour off/pipette off the pentane and transfer thematerial into a small lyophilization vessel. Place the vessel into avacuum oven at 37° C. for three hours (maximum vacuum) to extract theresidual pentane. Prepare a dry ice/pentane bath, snap freeze thematerial for 15 minutes, and then lyophilize to remove residual waterand solvent. Store appropriately.

Example 5

Prepare a DCM/Span 80 solution at 0.0045 g/mL, and a 3% Tween 80solution in type I water. Solubilize lidocaine HCL in the Tween solutionat the desired concentration. Prepare a 7.5% wt/v PLLA solution indichloromethane/Span 80 using high molecular weight polymer. Whencomplete, pipette 500 μL of the prepared lidocaine HCL/Tween 80 solutioninto the polymer solution to create a primary emulsion. Emulsifyaccording to standard practice, preferably using pulsed sonication. Loadthe emulsion into a 10 mL rubber-free syringe, attach a small diameterblunt needle, and deliver the contents at a rate of 0.150 mL/min, 5inches above a vessel containing approximately 600 mL of pentane. Thevessel also contains a filter screen or basket to capture formedparticles. When solution delivery is complete, wait 30 minutes to allowfor particle solidification, and then transfer the particles into alyophilization flask. Place the flask into a vacuum oven at 37° C. andallow the particles to dry for three hours. Load the particles into ascintillation vial or similar vessel, and immerse the vial into apentane/dry ice bath to freeze the material. Lyophilize for a minimum of24 hours to remove residual water and pentane.

Example 6

Prepare 30.0 ml of 0.4M AOT/isooctane volumetrically using a 50 mlcentrifuge tube. Create a solution of PEGylated alginate at 74 mg/ml inpure water. Prepare CaCl₂ solution at a concentration of 110 mM infiltered nanopure water. Dissolve BSA at a concentration of 7.33 mg/mlin filtered, nanopure water. Load 0.77 ml of PEGylated alginate solutioninto a 3 ml disposable syringe (A). Load 0.33 ml of BSA solution intoanother 3 ml disposable syringe (B), and load 0.11 ml of CaCl₂ solutioninto syringe a third syringe (C). Connect syringes A&B and mix the twosolutions by moving the material from one syringe to the other 20 times.Separate the two syringes and connect a new (D) 3 ml disposable syringeto the syringe containing the mixed material. Push the PEGylatedalginate/BSA solution into the new syringe through a 0.2 μm filter.Dispose of the empty syringe and filter. Empty the syringe containing offiltered PEGylated alginate into a 1.5 mL centrifuge tube. Withdraw1.000 ml from the Eppendorf, and pipette it into the vial containing thelyophilized protein to be loaded. Mix well, but do not vortex thematerial. Connect a sterile needle to syringe D and carefully withdrawthe fluid from the vial containing the protein. Connect syringe D tosyringe C, which contains CaCl₂ solution. Push the PEGylatedalginate/BSA/protein into the CaCl₂ solution, then syringe back andforth 20 times to ensure that the Ca²⁺ is well dispersed. Immediatelyempty the syringe contents into previously prepared 20.9 ml of 0.4MAOT/isooctane in a 50 ml centrifuge tube. Cap the tube and vortex. Placethe tube vertically in the refrigerator at 4° C. immediately aftervortexing. Allow to gel overnight. Lightly centrifuge the tube at 1500rpm for 15 minutes. Discard the supernatant without disturbing thepellet and then wash the retained material three times with ethanol.Remove/evaporate the ethanol and store the resulting material for use.

Example 7

Create a dextran/lysozyme solution in nanopure water, with dextran 70 at6% w/w, and with lysozyme added at 2 mg/mL. Create a PEG 8000 solutionat 6% w/w. Create a 1:10 w/w blend of the dextran 70/lysozyme:PEG 8000solutions and vortex to blend. These ratios should allow for thecreation of a single phase aqueous system. Place the solutions in thefreezer at −20° C. for at least 8 hours to slow freeze and phaseseparate into a water-in-water emulsion. Snap freeze and lyophilize thevial for 48 hours, minimum. When complete, add 10 mL dichloromethane tothe tube, vortex, and then centrifuge for 15 minutes to collect theformed dextran particles. Discard the supernatant, refill with DCM, andcentrifuge again. Repeat this washing process a total of three times.Dry the tubes for at least 8 hours in a vacuum oven at RT and maximumvacuum.

Example 8

As in the previous example, create a 1:10 w/w blend of dextran 70/PEG8000 solution containing a protein of interest. Spray atomize thesingle-phase solution into liquid nitrogen to create ice particles ofPEG/dextran/protein that are sub-25 μm. Collect the particles whilefrozen and disperse into a vessel of cyclohexane chilled to 7° C. Thefrozen dextran/PEG/protein particles should locally freeze thecyclohexane, which has a freezing temperature of approximately 6.5° C.,and remain dispersed. Immediately hard freeze the suspension in dryice/pentane, and then allow the frozen material to return to 4° C. tothaw the aqueous portion but maintain the cyclohexane in a frozen state.Hold at this temperature for an hour, and then transfer the material toa −20° C. freezer for at least 8 hours to invoke temperature-inducedphase separation of the dextran/PEG and vitrification of the dextran.The resultant glassy dextran will encapsulate and protect the protein.Hard freeze in dry ice/pentane, and then lyophilize to recoverparticles. Wash with DCM repeatedly to remove PEG.

Example 9

Create a solution of dextran 70 containing a protein of interest. Usinga sonic atomizer, spray atomize the solution into liquid nitrogen tocreate ice particles of dextran that are sub-20 μm. Collect theparticles while frozen and disperse into a vessel of cyclohexane chilledto 7° C. The frozen dextran/protein particles should locally freeze thecyclohexane, which has a freezing temperature of approximately 6.5° C.,and remain dispersed. Immediately hard freeze the suspension in dryice/pentane, and then allow the frozen material to return to 4° C. tothaw the aqueous portion but maintain the cyclohexane in a frozen state.Hold at this temperature for an hour, and then transfer the material toa −20° C. freezer for at least 8 hours to invoke temperature-inducedvitrification of the dextran. The resultant glassy dextran willencapsulate and protect the protein. Hard freeze in dry ice/pentane, andthen lyophilize to recover particles.

Example 10

Create a solution of dextran 70 at 10% w/vol in a scintillation vial andallow to solubilize at 37° C. for 30 minutes. Weigh trehalose into ascintillation vial to obtain a desired end concentration of 10% w/vol.Add an appropriate volume of the dextran solution to this vial and allowto solubilize. Measure Tween 80 into a scintillation vial to obtain thedesired final volume at a concentration of 5.8% wt/vol. Add thedextran/trehalose solution to this to obtain the desired volume ofaqueous solution. Prepare a Span 80/cycloheptane solution at 1.18%w/vol. To a vial containing 1 mg protein, add 500 μL of the preparedaqueous solution. Mix gently to incorporate. Pipette this solution intoa vial containing 7.5 mL of the cycloheptane/Span 80 solution andemulsify to create a nano-size colloidal suspension. Transfer theemulsion to a 20 mL lyophilization vial and place onto a shelf that hasbeen pre-cooled to −55° C. Allow to freeze for 2 hours. Lyophilize thematerial to recover the lyocake containing nanoparticles of proteinencapsulated in trehalose/dextran.

Without further elaboration, it is believed that one skilled in the artcan, using the description herein, utilize the present invention to itsfullest extent. The embodiments described herein are to be construed asillustrative and not as constraining the remainder of the disclosure inany way whatsoever. While the preferred embodiments have been shown anddescribed, many variations and modifications thereof can be made by oneskilled in the art without departing from the spirit and teachings ofthe invention. Accordingly, the scope of protection is not limited bythe description set out above, but is only limited by the claims,including all equivalents of the subject matter of the claims. Thedisclosures of all patents, patent applications and publications citedherein are hereby incorporated herein by reference, to the extent thatthey provide procedural or other details consistent with andsupplementary to those set forth herein.

What is claimed is:
 1. A method of forming particles containing apharmaceutical or biological agent that preserves biological activity ofthe pharmaceutical or biological agent when exposed to an organicsolvent from which protection is desired, the method comprising:preparing a solution, formed from water as a solvent, comprising: apharmaceutical or a biological agent; and a substrate that is soluble inthe solution, comprising one or more chemical species; combining thesolution with an oil phase to form a water-in-oil emulsion in which thesolution is dispersed in the oil phase; lyophilizing the emulsion;wherein the particles are formed prior to or simultaneously with thelyophilizing, wherein the pharmaceutical or biological agent isentrapped by the formed particles, wherein the substrate composition andthe oil phase are selected so that the particles formed are suspendablein the organic solvent from which protection is desired and thebiological activity of the pharmaceutical or biological agent ispreserved upon suspension of the particles in the organic solvent fromwhich protection is desired, and wherein one or more substances selectedfrom the group consisting of a surfactant, a stabilizer, an emulsifier,and combinations thereof are incorporated as part of the substrate priorto solidification in order to form the emulsion.
 2. The method accordingto claim 1, wherein the emulsion further comprises lyoprotectants,cryoprotectants, or combinations thereof.
 3. The method according toclaim 1, wherein the substrate comprises one or more of the following: acarbohydrate selected from the group consisting of a polysaccharide, amonosaccharide, a disaccharide, and an oligosaccharide; a surfactant;and an emulsifier.
 4. The method according to claim 1, wherein theparticles have diameters that are less than 1 μm.
 5. The methodaccording to claim 1 wherein the oil phase comprises a solvent selectedfrom the group consisting of cyclohexane and cycloheptane.
 6. The methodaccording to claim 1 wherein chemical cross-linking causes thesolidification of the substrate.
 7. The method according to claim 1wherein physical cross-linking causes the solidification of thesubstrate.
 8. The method according to claim 1, wherein thesolidification process causes vitrification of the substrate.
 9. Themethod according to claim 1, wherein the solidification process causescrystallization of the substrate.